Human Genome Project: Overview and Impact

• Polymerase Chain Reaction

The Human Genome Project

• Started in 1990, involving about 30 different institutions

Human Genome Project Goal

• Fully sequence the human genome
• Sequencing of chemical bases that form human DNA
• Identify all genes in the human genome
• Development of tools for genetic information analysis

Importance of the Human Genome Project

• Helped scientists understand the development, prevention, and treatment of diseases
• Resulted in new technologies and analytical tools
• Established an open approach to data sharing
• Advanced sharing of scientific data policies and support
• Increased awareness of ethical concerns

Impact of the Project on Medicine

• Helped establish somatic cell genetic disease as a genetic disease category
• Define mutation as the base of cancer
• Altered the medicine practice

Impact on Other Fields

• Inspired data acquisition initiatives, like the International HapMap Project and The Cancer Genome Atlas
• Initial completion in April 2003
• Mission accomplished in May 2021
• Total size of ~3.2 billion bp.

Issues with Studying Specific DNA Sequences

• There are other sequences in a genome that are not of interest (Specificity)
• The amount of DNA in interested samples is very small (Amplification)
• Inefficiency in studying specific sequences
• PCR enables studying specific sequences

Kary Mullis

• Developed a way to double test target DNA by providing 21 copies
• Repeating N times would yield: 2N
• Adds an oligonucleotide primer with the 4 dNTPs and DNA polymerase in a chain reaction
• If N = 30 or 40, one would have millions of copies
• Won the 1993 Nobel Prize in Chemistry for discovering a way to analyze DNA easily and cheaply
• Paved the way for advancements in molecular biology, medical diagnostics, and forensic science

Polymerase Chain Reaction (PCR) Steps

• Molecular biology technique used to amplify DNA

Template DNA (Single Copy)

• Process begins with a single DNA strand functioning as the amplification template

PCR Cycles - First Cycle

• Denaturation: Double-stranded DNA is heated to separate into single strands
• Annealing: Primers attach to complementary sequences on the single-stranded DNA
• Extension: DNA polymerase synthesizes new DNA strands using the primers as starting points
• After the first cycle, 2 copies of the original DNA are produced

PCR Cycles - Second Cycle

• The newly synthesized DNA strands act as templates for another round of PCR
• The process repeats, leading to 4 copies of the target DNA sequence
• Third cycle repeats, producing 8 copies
• DNA strands accumulate exponentially
• The process continues for multiple cycles of around 30 cycles in a standard PCR reaction
• By the 30th cycle, approximately copies of the target DNA sequence are generated, showing the PCR's DNA amplification power
• PCR achieves exponential DNA replication through denaturation, annealing, and extension repeated cycles

Denaturation

• Denaturation defines the unfolding or breaking up of proteins, modifying its standard three-dimensional structure
• Proteins can be denatured by agitation, heat, or chemical action, causing protein unfolding, leaving molecules non-functional
• Double-stranded DNA is separated into 2 single strands
• Temperature of 94-96°C
• Time: Several seconds to minutes
• Larger template = longer time

Annealing

• Most critical step for specificity
• Primers hybridize to their complementary DNA sequences
• Temperature: 50-70°C
• Starting point is determined using the Tm of the primer sequences

Melting Temperature (Tm)

• Tm, or melting temperature, in PCR is used to control the temperature at which the primers attach to DNA
• It is a critical metric in PCR and other molecular biology experiments
• Why is Tm important?
• During Annealing:
• The reaction temperature needs to be low enough for primers to bind to the DNA template
• High enough to prevent the formation of undesired duplexes
• During Amplification:
• All primers in a reaction should have similar Tm values so they can dissociate and anneal from DNA sequences at similar temperatures
• Enables each amplification to proceed at the selected temperature
• During Sequence Verification:
• Melting analysis following PCR monitors duplex hybridization as the temperature changes
• Simple method for genotyping and sequence verification

Tm Range

• Melting temperature (Tm) range of 52-58°C generally produces the best results
• Primers with a Tm above 65°C leads to secondary annealing.

DNA Strands

• 5'-3' leading strand
• 3'-5' lagging strand
• The 5' to 3' leading strand is a DNA strand synthesized continuously
• The 3' to 5' lagging strand is synthesized in small, discontinuous fragments called Okazaki fragments

Extension

• Performed by DNA polymerase
• Polymerase synthesizes a template DNA copy
• Catalyzes the phosphodiester bonds formation between dNTPs and the primer's 3' end
• Temperature: 68-72°C
• End result: The template number doubles

Taq Polymerase

• Synthesizes new DNA strands by adding nucleotides to the template during the extension phase
• Heat-resistant nature is crucial for PCR amplification
• A heat-stable DNA polymerase enzyme extracted from the bacteria "Thermus aquaticus"

PCR Components

• Primers
• Single-stranded DNA fragments, 20-30 bp long
• Works like the primers in vivo
• Determine the PCR's specificity
• Contain sites flanking the interested region with sequences that are complementary
• Forward Primer
• 5' to the amplified sequences
• Hybridizes with the minus strand
• Reverse Primer
• 3' to the amplified sequences
• Hybridizes with the plus strand

DNA Polymerase

• Forwards will create a copy that is that will mirror the target gene
• Primer sequence (%GC) helps bind to the target
• Length of strand
• The melting temperature (Tm) of the forward and reverse primer should be similar

DNA Template

• From nucleotide extraction
• Routine clinical analyses require 100 ng – 1 μg
• The best templates are in good condition, free of contaminants and free of nicks and breaks

Deoxyribonucleotide Bases

• Building blocks of DNA
• dNTPs
• dATP (adenine)
• dTTP (thymine)
• dGTP (guanine)
• dCTP (cytosine)
• Usual requirement: 0.1-0.5 mM of each
• The first polymerase from E. coli
• First polymerase to be added after each denaturation round

Taq Polymerase Types

• Thermus aquaticus Thermal stable Good fidelity Fidelity = Accurate copying of the template

Thermus aquaticus Species

• Bacteria species that tolerates elevated temperatures
• One of several thermophilic bacteria that belong thermophilic bacteria that belong to the Deinococccota phylum

Thermus aquaticus Taq Polymerase function

Belongs to the amplification stage of a PCR reaction, as it is the enzyme responsible for synthesizing new DNA strands Addition of nucleotides occurs on extension Heat-resistant nature is crucial for PCR amplification.

• The Taq polymerase is a heat-stable DNA enzyme, derived from "Thermus aquaticus"
• Commonly used in PCR to repeatedly copy specific DNA-sequences
• Its’ ability to copying at elevated-temperatures with no denaturing allows various cycles of DNA in a single cycle

Tth Polymerase qualities

• Derived From Thermus thermophilus
• Also has reverse transcriptase activity
• Used in RT-PCR when using an RNA template Is-a Gram-negative bacterium, utilized during biotechnological applications,
• Functions as a model during genetic manipulation, genomes structuration as well as systems-biology

Functions of PR Buffers

• Provide favorable conditions for ideal enzyme activity
• Composed of pH buffers as well as salts that give the cations needed
• During buffer solutions it functions by: Salt stabilizing pH, aiding PCR reagent experiments

PCR Buffer - Purpose

• Maintains PH by neutralizing any acidic or basic compounds in the sample
• It also promotes enzyme activity by including potassium or ammonium ions for optimal primer binding with the template
• Improves PCR specificity, as well as stabilizes improper binding for off-target locations

Buffer reagents

Includes: Salts such as: (Magnesium chlorine, potassium chloride. as well as tris(hydroxymethyl)aminomethane (Tris)) Stabilizers together with enhancing agents such as: (Gelatin or bovine serum albumin/BSA) Taq polymerase:( Can be premixed alongside the buffer to limit steps

TRIS buffers

• Tris Buffers, provides an ideal activity in means of PH; and precise/ accurate enhancement
• Provides pH for optimal enzyme activity and accurate amplification PH 8 -9.5 Can have additional parts.

Supplemental portions of Tris

• Albumin-Bovine serum: ( ranging 10-100 μg/mL) ( this removes hinderances, and will stabilize enzymes)
• Dithiothreitol( a range of 0.01mM ) ( gives less conditions, improving with enzymes)
• The denaturing temperatures of DNA will be lowered by using : Formamide, around (1%-10%) This may increase opportunity/ availability in the primers
• As well as diminish structuration to allow polymerase’s full increase through problematic/ difficult.
• In the use of Triton X-100 as well as Dimethyl sulfoxide and glycerol can range ( 1%- 10%)
• Monovalent Cations- (KCl) when using (NH4)2SO4
  • Alters by both denaturing and also annealing’/ increases
  • Increasing concentrations means longer arrangement in lowering DNA arrangement

PCR mono valent and Divalent

• Divalent Cations- MgCI2 = Is a requirement by the DNA pol
• During 1 NTP = it needs up to 1 Mg atom
• If numbers are low then it effects - By EDTA/other chelators
• • ↓ amplicons meaning low products may occur
• when level are to high then
• • ↑ Nonspecific Product may be the result

Oligodeoxynucleotide

• Short DNA chain/ Molecule used for different processes, such as therapeutic and or genetic testing reason or moleculars research
• and Can be composed of: 0.25 mM with each primer utilized,(With usage of (oligodeoxynucleotides))
• composed of 0.2 with each dATP, dCTP, dGTP, dTTP
• -Can range up to. is, or range from; 50.
• mM with KCI/as well as 10mM Tris, pH 8.4/as well as 1.5 mM MgCl₂, = with ranges 2.5 units
• -Poly with several copies of the template like ;102-105 copies

ODN Composition

Molecules/Chains Made - in a lab In a process called: solid phase synthesis 4 Nucleic Are needed Chains Formed - in various nucleotides

Thermal Cyclers used During different types of Reactions/Processes

1st =multiple heat water baths

• Manually-operated Rapid-Changes needed for automatic runs- Rapid
• Used During: designated- periods Cycles formed by temperatures- that are programmed =
  • PCR( polymerase Chain reactions) = Used With Thermal( cycling This helps copy fragments that are specified helping reactions such as; Help different phases like: ( denaturation as well as temp with extra steps)

Functionality/ components of Applications:

A heating black with holes utilized= contains PCR tubes needed for mixing various reactions needed Used for processes like:

• DNA structured/sequenced as well as used with cloning to enhance genetic mutagen, And to help: amplify DNA

Positive Control:

Used During

• Active enzyme Best Buffer Used
• Good/Optimal Primers with Primer During Mech Runs (REAGENT BLANK) Non Effect
• Without DNA Negative Control
• To Help Contaminants (TEMPLATE/Negative Lacks Targeted Structs

Contaminants in different PCR

Balance is Needed/ aggressive Amplification The contamination is at times Major Products form Resolved PHYSICALLY/ or In some cases chemically used

Physical and/ or used through Separation to Prevent Issues can

Preventing- (contamination Damages formed Single structures break

Chemicals used

Reactions

• UV lighting Damage Breaks both single/ or structured Enhance efficiency psoralens

Types used

(Interfere/or prevent= amplification by DNA Help Prevent Structs. Bleach ( 10%)

• A widely used method for workspace clean up as well as decontamination -Hypochlorite solutions = at least ranging 7mM helps, or at most specimens’ touching, to eliminate almost any contamination from any DNA forms = used systemically

Primers

Replace

• Substituted
• UNG - degradation

Primer Functions- for the mis functioned and are not working Binds poorly to DNA

• Primes used badly - causes for future Bad DNA formations Primers are bad/binding badly Causes
• Polymerase Bad - causes for actions at room( high temp ) Causes strands to have: low stringent issues (Solution good to help prepare is, and helps in Good strands design)
• During optimal reactions

Solvents

• Harmful & Not Effective

The cycles occur Help reaction RT/ Pcr Helps lower temps- as well

Reaction that can Occur and used during specific cases can include: Primers with many rx that can use strands can result in many many go. But can also result in inefficient rx

Pcr Modifications

Used to amplifications, with singular targets in separate programs Advantage Increases sensitiveness and precise Disadvantage - is at times Time consuming for processes with many steps during -the usage of start up for RNA Templates To many samples, there are many to use and have

Real-time PCR:

Can Find High DNA, if/or when

• It’s there, with fluorescent methods The different- types, for assays When the primers are good to use for (Strands/ Assays) good.
• And, they are, not toxic with no effect - During the cycles for reactions

Assay used to find is

• Replaced EtBr
• specific
• Cheap as a result And High During: tests